Nucleotide sequences which code for the ccpa2 gene

ABSTRACT

The invention relates to polynucleotides corresponding to the ccpA2 gene and which encode a CcpA2 catabolite control protein, methods of producing L-amino acids, and methods of screening for polynucleotides which encode proteins having CcpA2 catabolite control activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to German Application No.DE 10042053.2 filed Aug. 26, 2000 and German Application No. DE10123071.0 filed May 11, 2001, the entire contents of both applicationsare incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention provides nucleotide sequences from Coryneformbacteria which code for the ccpA2 gene and a process for thefermentative preparation of amino acids, in particular L-lysine, byattenuation of the ccpA2 gene. The ccpA2 gene codes for the CcpA2protein, which is the catabolite control protein A.

[0004] 2. Discussion of the Background

[0005] L-Amino acids, particularly L-lysine, are used in human medicineand in the pharmaceuticals industry, in the foodstuffs industry and,most particularly, in animal nutrition.

[0006] It is known that amino acids are prepared by fermentation ofstrains of Coryneform bacteria, in particular Corynebacteriumglutamicum. Because of their great importance, attempts are continuouslybeing made to improve the preparation processes. Improvements to theprocess may concern measures relating to fermentation, for example,stirring and oxygen supply, or the composition of the nutrient media,such as the sugar concentration during the fermentation, or the workingup to the product form by, for example, ion exchange chromatography, orthe intrinsic output properties of the microorganism itself.

[0007] The output properties of these microorganisms are improved byemploying methods of mutagenesis, selection and mutant selection. Thesemethods yield strains that produce amino acids and are resistant toantimetabolites or are auxotrophic for metabolites important forregulation.

[0008] For a number of years, methods of recombinant DNA technology havealso been used for improving the L-amino acid-producing strains ofCorynebacterium. However, there remains a critical need for improvedmethods of producing L-amino acids and thus for the provision of strainsof bacteria producing higher amounts of L-amino acids. On a commercialor industrial scale even small improvements in the yield of L-aminoacids, or the efficiency of their production, are economicallysignificant. Prior to the present invention, it was not recognized thatattenuation of the ccpA2 gene encoding the catabolite control protein A(CcpA2) would improve L-amino acid yields.

SUMMARY OF THE INVENTION

[0009] An object of the present invention is to provide novel measuresfor the improved production of L-amino acids or amino acid, where theseamino acids include L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan, L-arginine and the salts (monohydrochloride or sulfate)thereof.

[0010] One object of the present invention is providing a novel processfor improving the fermentative production of said L-amino acids,particularly L-lysine. Such a process includes enhanced bacteria,preferably enhanced Coryneform bacteria, which express attenuatedamounts of the CcpA2 catabolite control activity.

[0011] Thus, another object of the present invention is providing such abacterium, which expresses an attenuated amount of CcpA2 catabolitecontrol protein or gene products of the ccpA2 gene.

[0012] Another object of the present invention is providing a bacterium,preferably a Coryneform bacterium, which expresses a polypeptide thathas an attenuated CcpA2 catabolite control activity.

[0013] Another object of the invention is to provide a nucleotidesequence encoding a polypeptide which has CcpA2 catabolite controlprotein sequence. One embodiment of such a sequence is the nucleotidesequence of SEQ ID NO: 1.

[0014] A further object of the invention is a method of making CcpA2catabolite control protein or an isolated polypeptide having a CcpA2catabolite control activity, as well as use of such isolatedpolypeptides in the production of amino acids. One embodiment of such apolypeptide is the polypeptide having the amino acid sequence of SEQ IDNO: 2.

[0015] Other objects of the invention include methods of detectingnucleic acid sequences homologous to SEQ ID NO: 1, particularly nucleicacid sequences encoding polypeptides that have CcpA2 catabolite controlactivity, and methods of making nucleic acids encoding suchpolypeptides.

[0016] The above objects highlight certain aspects of the invention.Additional objects, aspects and embodiments of the invention are foundin the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0017]FIG. 1. Map of the plasmid pCR2.1ccpA2int.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art of molecular biology. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

[0019] Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1982) and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989) and the various references cited therein.

[0020] The invention provides an isolated polynucleotide from Coryneformbacteria, containing a polynucleotide sequence coding for the ccpA2gene, selected from the group consisting of

[0021] a) polynucleotide that is at least 70% identical to apolynucleotide that codes for a polypeptide containing the amino acidsequence of SEQ ID No. 2,

[0022] b) polynucleotide that codes for a polypeptide containing anamino acid sequence that is at least 70% identical to the amino acidsequence of SEQ ID No. 2,

[0023] c) polynucleotide that is complementary to the polynucleotides ofa) or b), and

[0024] d) polynucleotide containing at least 15 successive nucleotidesof the polynucleotide sequence of a), b) or c),

[0025] the polypeptide preferably having the activity of the catabolitecontrol protein CcpA2.

[0026] The invention also provides the above-mentioned polynucleotide,preferably being a replicatable DNA containing:

[0027] (i) the nucleotide sequence shown in SEQ ID No. 1 or

[0028] (ii) at least one sequence that corresponds to sequence (i)within the range of the degeneracy of the genetic code, or

[0029] (iii) at least one sequence that hybridizes with the sequencesthat are complementary to sequences (i) or (ii), and optionally

[0030] (iv) sense mutations in (i) that are neutral in terms offunction.

[0031] The invention also provides:

[0032] a replicatable DNA containing the nucleotide sequence as shown inSEQ ID No.1;

[0033] a polynucleotide that codes for a polypeptide containing theamino acid sequence as shown in SEQ ID No. 2;

[0034] a vector containing parts of the polynucleotide according to theinvention, but at least 15 successive nucleotides (point d, supra),particularly pCR2.1ccpA2int, deposited in Escherichia coli DSM 14257 atthe DSMZ, Braunschweig (Germany);

[0035] and Coryneform bacteria that contain in the ccpA2 gene aninsertion or deletion, particularly using the vector pCR2.1ccpA2int.

[0036] The invention also provides polynucleotides consistingsubstantially of a polynucleotide sequence, which are obtainable byscreening, by means of hybridization, of a corresponding Coryneform genelibrary that contains the complete gene having the polynucleotidesequence according to SEQ ID No. 1, using a probe containing thesequence of said polynucleotide according to SEQ ID No.1 or a fragmentthereof, and isolating said polynucleotide sequence.

[0037] Polynucleotide sequences according to the invention are suitableas hybridization probes for RNA, cDNA and DNA, in order to isolate thefull length nucleic acids or polynucleotides or genes that code for theCcpA2 protein, or in order to isolate nucleic acids or polynucleotidesor genes that have a high similarity with the sequence of the ccpA2gene.

[0038] The DNA of genes that code for the CcpA2 protein can be preparedwith the polymerase chain reaction (PCR) by using the polynucleotidesequences according to the invention as primers.

[0039] Such oligonucleotides acting as probes or primers contain atleast 30, preferably at least 20, more preferably at least 15,consecutive nucleotides. Also suitable are oligonucleotides that have alength of at least 40 or 50 nucleotides.

[0040] “Isolated” means removed from its natural environment.

[0041] “Polynucleotide” generally refers to polyribonucleotides andpolydeoxyribonucleotides. The RNA or DNA may be modified or unmodified.

[0042] The polynucleotides according to the invention include apolynucleotide shown in SEQ ID No. 1 or a fragment prepared therefromand also those that are at least 70%, preferably at least 80% and inparticular at least 90% to 95% identical to the polynucleotide accordingto SEQ ID No. 1 or a fragment prepared therefrom.

[0043] “Polypeptides” are understood as being peptides or proteins thatcomprise two or more amino acids bonded via peptide bonds.

[0044] The polypeptides according to the invention include a polypeptideshown in SEQ ID No. 2 particularly those having the biological activityof the CcpA2 protein, and also those that are at least 70%, preferablyat least 80% and in particular at least 90% to 95% identical with thepolypeptide shown in SEQ ID No. 2 and exhibit the mentioned activity.

[0045] The invention also provides a process for the production of aminoacids, particularly L-lysine, by fermentation using Coryneform bacteriawhich, in particular, already produce amino acids and in which thenucleotide sequences coding for the ccpA2 gene are attenuated, inparticular excluded or expressed at a low level.

[0046] The term “attenuation” in this connection describes the reductionor exclusion of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,by, for example, using a weak promoter or using a gene or allele thatcodes for a corresponding enzyme with a low activity, or by inactivatingthe corresponding gene or enzyme (protein), and optionally by combiningthose measures. As a result of attenuation, the activity orconcentration of the corresponding protein is, in general, reduced to 0to 50%, 0 to 25%, 0 to 10%, or 0 to 5% of the wild-type protein activityor concetration.

[0047] The term “enhancement” in this connection describes the increasein the intracellular activity of one or more enzymes in a microorganismwhich are coded by the corresponding DNA, by, for example, increasingthe number of copies of the gene or genes, using a potent promoter orusing a gene which codes for a corresponding enzyme having a highactivity, and optionally combining those measures. As a result ofenhancement, in particular over-expression, the activity orconcentration of the corresponding protein is increased, in general,preferably ranging from at least 10%, 25%, 50%, 75%,100%,150%,200%,300%, 400%, or 500%, up to 1000% or 2000% of the wild-typeprotein activity or concentration present in the microorgansim.

[0048] The microorganisms provided by the present invention can prepareamino acids, in particular L-lysine, from glucose, sucrose, lactose,fructose, maltose, molasses, starch, cellulose or from glycerol andethanol. The microorganisms can be representatives of Coryneformbacteria, in particular of the genus Corynebacterium. Corynebacteriumglutamicum species of this genus garners special mention since it iswell known to those skilled in the art for its ability to produceL-amino acids.

[0049] Suitable strains of the genus Corynebacterium, particularly thespecies Corynebacterium glutamicum (C. glutamicum), are, in particular,the known wild-type strains

[0050]Corynebacterium glutamicum ATCC13032

[0051]Corynebacterium acetoglutamicum ATCC15806

[0052]Corynebacterium acetoacidophilum ATCC13870

[0053]Corynebacterium melassecola ATCC17965,

[0054]Corynebacterium thermoaminogenes FERM BP-1539

[0055]Brevibacterium flavum ATCC 14067

[0056]Brevibacterium lactofermentum ATCC13869 and

[0057]Brevibacterium divaricatum ATCC 14020

[0058] or L-amino acid-producing mutants or strains prepared therefrom,for example, the L-lysine-producing strains

[0059]Corynebacterium glutamicum FERM-P 1709

[0060]Corynebacterium glutamicum FERM-P 6463

[0061]Corynebacterium glutamicum FERM-P 6464

[0062]Corynebacterium glutamicum DM58-1

[0063]Corynebacterium glutamicum DG52-5

[0064]Corynebacterium glutamicum DSM 5715

[0065]Corynebacterium glutamicum DSM 12866

[0066]Brevibacterium flavum FERM-P 1708 and

[0067]Brevibacterium lactofermentum FERM-P 1712

[0068] Preferably, a bacterial strain with attenuated expression of accpA2 gene that encodes a polypeptide with CcpA2 activity will improveamino acid yield at least 1%.

[0069] The inventors have succeeded in isolating the new ccpA2 gene ofC. glutamicum that codes for the CcpA2 protein, which is a catabolitecontrol protein A.

[0070] To isolate the ccpA2 gene or also other genes of C. glutamicum, agene library of that microorganism is first prepared in Escherichia coli(E. coli). The preparation of gene libraries is described in generallyknown textbooks and handbooks. For example, the textbook of Winnacker:Gene und Klone, Eine Einführung in die Gentechnologie (Verlag Chemie,Weinheim, Germany, 1990) or the handbook by Sambrook et al.: MolecularCloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press,1989). A well-known gene library is that of the E. coli K-12 strainW3110, which has been prepared by Kohara et al. (Cell 50, 495-508(1987)) in λ vectors. Bathe et al. (Molecular and General Genetics,252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032,which was prepared with the aid of the cosmid vector SuperCos 1 (Wahl etal., 1987, Proceedings of the National Academy of Sciences, USA,84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988,Nucleic Acids Research 16:1563-1575). Börmann et al. (MolecularMicrobiology 6(3), 317-326)) (1992)) in turn describe a gene library ofC. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, 1980,Gene 11, 291-298).

[0071] It is possible to use plasmids such as pBR322 (Bolivar, 1979,Life Sciences, 25, 807-818) or pUC9 (Vieira et al., 1982, Gene,19:259-268) in order to prepare a gene library of C. glutamicum in E.coli. Suitable hosts are particularly those E. coli strains, which arerestriction- and recombination-deficient, such as, the DH5α strain(Jeffrey H. Miller: “A Short Course in Bacterial Genetics, A LaboratoryManual and Handbook for Escherichia coli and Related Bacteria”, ColdSpring Harbour Laboratory Press, 1992).

[0072] The long DNA fragments cloned with the aid of cosmids or other λvectors can then be subcloned in turn into the usual vectors suitablefor DNA sequencing.

[0073] Methods of DNA sequencing are described inter alia by Sanger etal. (Proceedings of the National Academy of Sciences of the UnitedStates of America USA, 74:5463-5467, (1977).

[0074] The resulting DNA sequences can then be studied using knownalgorithms or sequence analysis programs, such as that of Staden(Nucleic Acids Research 14, 217-232 (1986)), that of Marck (NucleicAcids Research 16, 1829-1836 (1988)) or the GCG program of Butler(Methods of Biochemical Analysis 39, 74-97 (1998)).

[0075] In that manner, the novel DNA sequence of C. glutamicum whichcodes for the ccpA2 gene (SEQ ID No. 1) has been obtained and forms partof this invention. Furthermore, the amino acid sequence of thecorresponding protein has been derived from the present DNA sequence bythe methods described above. The resulting amino acid sequence of theccpA2 gene product is shown in SEQ ID No. 2.

[0076] Coding DNA sequences that result from SEQ ID No. 1 by thedegeneracy of the genetic code also form part of the invention. In thesame way, DNA sequences which hybridize with SEQ ID No. 1 or parts ofSEQ ID No. 1 form part of the invention. Furthermore, to a personskilled in the art, conservative amino acid exchanges, such as exchangeof glycine for alanine or of aspartic acid for glutamic acid inproteins, are known as “sense mutations.” These mutations do not lead toa fundamental change in the activity of the protein, i.e. are neutral interms of function. It is also known that changes at the N and/or Cterminus of a protein may not substantially impair or may even stabilizethe function thereof. The person skilled in the art will find relevantinformation inter alia in Ben-Bassat et al. (Journal of Bacteriology169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), inSahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al.(Bio/Technology 6:1321-1325 (1988)) and in known textbooks of geneticsand molecular biology. Amino acid sequences that result in acorresponding manner from SEQ ID No. 2 also form part of the invention.

[0077] Finally, DNA sequences, which are prepared by the polymerasechain reaction (PCR) using primers that result from SEQ ID No. 1 formpart of the invention. Such oligonucleotides typically have a length ofat least 15 nucleotides.

[0078] A person skilled in the art will find instructions foridentifying DNA sequences by means of hybridization inter alia in thehandbook “The DIG System Users Guide for Filter Hybridization” fromBoehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al.(International Journal of Systematic Bacteriology 41: 255-260 (1991)).Hybridization takes place under stringent conditions, that is to sayonly hybrids in which the probe and target sequence, i.e. thepolynucleotides treated with the probe, are at least 70% identical areformed. It is known that the stringency of the hybridization, includingthe washing steps, is influenced or determined by varying the buffercomposition, the temperature and the salt concentration. For reasonsexplained infra, the hybridization reaction is preferably carried outunder a relatively low stringency compared with the washing steps(Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

[0079] A 5×SSC buffer at a temperature of approximately 50-68° C., forexample, can be employed for the hybridization reaction. Probes can alsohybridize here with polynucleotides which are less than 70% identical tothe sequence of the probe. Such hybrids are less stable and are removedby washing under stringent conditions. This can be achieved, forexample, by lowering the salt concentration to 2×SSC and subsequently0.5×SSC (The DIG System User's Guide for Filter Hybridisation,Boehringer Mannheim, Mannheim, Germany, 1995) with a temperature ofapproximately 50-68° C. being established. It is optionally possible tolower the salt concentration to 0.1×SSC. Polynucleotide fragments whichare, for example, at least 70% or at least 80% or at least 90% to 95%identical to the sequence of the probe employed can be isolated byincreasing the hybridization temperature stepwise in approximately 1-2°C. increments. Commercial kits containing further instructions onhybridization are readily obtainable (e.g. DIG Easy Hyb from RocheDiagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558).

[0080] A person skilled in the art will find instructions foramplification of DNA sequences with the aid of the polymerase chainreaction (PCR) inter alia in the handbook by Gait: OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and inNewton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg,Germany, 1994).

[0081] During work on the present invention it was found that Coryneformbacteria produce amino acids, in particular L-lysine, in an improvedmanner after attenuation of the ccpA2 gene.

[0082] To achieve an attenuation, either the expression of the ccpA2gene or the catalytic properties of the enzyme protein may be diminishedor excluded. The two measures may optionally be combined.

[0083] The reduction of gene expression may be effected by carrying outthe culturing in a suitable manner or by genetic modification (mutation)of the signal structures of gene expression. Signal structures of geneexpression are, for example, repressor genes, activator genes,operators, promoters, attenuators, ribosome binding sites, the startcodon and terminators. The person skilled in the art will findinformation on this in the patent application WO 96/15246, in Boyd andMurphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil andChambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer(Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al.(Microbiology 142: 1297 (1996)), Vasicova et al. (Journal ofBacteriology 181: 6188 (1999)) and in known textbooks of genetics andmolecular biology, such as the textbook by Knippers (“MolekulareGenetik”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) orthat by Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim,Germany, 1990).

[0084] Mutations that lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art; examplesthat may be mentioned are the works by Qiu and Goodman (Journal ofBiological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (BioscienceBiotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“DieThreonindehydratase aus Corynebacterium glutamicum: Aufhebung derallosterischen Regulation und Struktur des Enzyms”, Reports from theJülich Research Centre, Jül-2906, ISSN09442952, Jülich, Germany, 1994).Summaries are found in known textbooks of genetics and molecularbiology, such as that by Hagemann (“Allgemeine Genetik”, Gustav FischerVerlag, Stuttgart, 1986).

[0085] These mutations may be transitions, transversions, insertions anddeletions. Depending on the effect of the amino acid exchange on theenzyme activity, “missense mutations” or “nonsense mutations” arereferred to. Insertions or deletions of at least one base pair (bp) in agene lead to frame shift mutations, as a consequence incorrect aminoacids are incorporated or translation is interrupted prematurely.Deletions of several codons typically lead to a complete loss of theenzyme activity. Instructions on the production of such mutations arepart of the prior art and can be found in known textbooks of geneticsand molecular biology, such as the textbook by. Knippers (“MolekulareGenetik”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995),that by Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim,Germany, 1990) or that by Hagemann (“Allgemeine Genetik”, Gustav FischerVerlag, Stuttgart, 1986).

[0086] A common method of mutating genes of C. glutamicum is the methodof gene disruption and gene replacement described by Schwarzer andPühler (Bio/Technology 9, 84-87 (1991)).

[0087] In the gene disruption method, a central part of the codingregion of the gene of interest is cloned in a plasmid vector that isable to replicate in a host (typically E. coli), but not in C.glutamicum. Suitable vectors are, for example, pSUP301 (Simon et al.,Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al.,Gene 145, 69-73 (1994)), pK18mobsacB or pK19mobsacB (Jäger et al.,Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promegacorporation, Madison, Wisc., USA), pCR2.1-TOPO (Shuman Journal ofBiological Chemistry 269:32678-84 (1994); U.S. Pat. No. 5,487,993),pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal ofMolecular Biology, 234: 534-541 (1993)) or pEM1(Schrumpfet al, 1991,Journal of Bacteriology 173:4510-4516). The plasmid vector containingthe central part of the coding region of the gene is then transferredinto the desired strain of C. glutamicum by conjugation ortransformation. The method for conjugation is described, for example, bySchäfer et al. (Applied and Environmental Microbiology 60, 756-759(1994)). Methods for transformation are described by Thierbach et al.(Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican andShivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)) . After homologousrecombination by means of a cross-over event, the coding region of thegene in question is interrupted by the vector sequence and twoincomplete alleles are obtained, one lacking the 3′ end and one lackingthe 5′ end. This method has been used by Fitzpatrick et al. (AppliedMicrobiology and Biotechnology 42, 575-580 (1994)) to exclude the recAgene of C. glutamicum.

[0088] In the gene replacement method, a mutation, such as a deletion,insertion or base exchange, is established in vitro in the gene ofinterest. The allele prepared is in turn cloned in a vector that is notreplicated in C. glutamicum and this is then transferred into thedesired host of C. glutamicum by transformation or conjugation. Afterhomologous recombination by means of a first cross-over event effectingintegration and by means of a suitable second cross-over event effectingan excision in the target gene or in the target sequence, incorporationof the mutation or of the allele is achieved. This method was used byPeters-Wendisch et al.(Microbiology 144, 915-927 (1998)) to exclude thepyc gene of C. glutamicum by a deletion event.

[0089] A deletion, insertion or a base exchange can be incorporated intothe ccpA2 gene in this manner.

[0090] In addition, it may be advantageous for the production of L-aminoacids, in particular L-lysine, in addition to attenuation of the ccpA2gene, to amplify, in particular to over-express, one or more enzymes ofthe particular biosynthesis pathway, of glycolysis, of anaplerosis, ofthe citric acid cycle, of the pentose phosphate cycle or of amino acidexport and optionally regulatory proteins,.

[0091] Thus, for example, the preparation of L-lysine, one or more ofthe genes chosen from the group

[0092] the lysC gene which codes for a feed-back resistant aspartatekinase (Accession No.P26512; EP-B-0387527; EP-A-0699759),

[0093] the dapA gene which codes for dihydrodipicolinate synthase (EP-B0 197 335),

[0094] the eno gene which codes for enolase (DE: 19947791.4),

[0095] the zwf gene which codes for the zwf gene product(JP-A-09224661),

[0096] the dapD gene which codes for tetradihydrodipicolinatesuccinylase (Wehrmann et al., Journal of Bacteriology 180, 3159-3165(1998)),

[0097] the dapE gene which codes for succinyldiaminopimelatedesuccinylase (Wehrmann et al., Journal of Bacteriology 177: 5991-5993(1995)),

[0098] the gap gene which codes for glyceraldehyde 3-phosphatedehydrogenase (Eikmanns (1992). Journal of Bacteriology 174:6076-6086),

[0099] the pyc gene which codes for pyruvate carboxylase(Peters-Wendisch et al.(Microbiology 144, 915-927 (1998))

[0100] the mqo gene which codes for malate:quinone oxidoreductase(Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),

[0101] the zwal gene which codes for the Zwal protein (DE: 19959328.0,DSM 13115)

[0102] the lysE gene which codes for lysine export (DE-A-195 48 222)

[0103] may at the same time be enhanced, in particular over-expressed.

[0104] It may also be advantageous for the production of amino acids, inparticular L-lysine, in addition to the attenuation of the ccpA2 gene,at the same time to attenuate one or more of the genes chosen from thegroup

[0105] the pck gene which codes for phosphoenol pyruvate carboxykinase(DE 199 50 409.1, DSM 13047),

[0106] the pgi gene which codes for glucose 6-phosphate isomerase(U.S.Ser. No. 09/396,478, DSM 12969),

[0107] the poxB gene which codes for pyruvate oxidase (DE:1995 1975.7,DSM 13114),

[0108] the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2,DSM 13113).

[0109] It may also be advantageous for the production of amino acids,particularly L-lysine, in addition to attenuation of the ccpA2 gene toeliminate undesirable side reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

[0110] The microorganisms prepared according to the invention, for thepurpose of production of L-amino acids, in particular L-lysine, can becultured by batch process (continuous or discontinuous), fed batch, orrepeated fed batch process. A summary of known culture methods isdescribed in the textbook by Chmiel (Bioprozesstechnik 1. Einführung indie Bioverfahrenstechnik(Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen(ViewegVerlag, Braunschweig/Wiesbaden, 1994)).

[0111] A suitable culture medium must be used to meet the requirementsof the particular strains. Descriptions of culture media for variousmicroorganisms are found in the handbook “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981). Sugars and carbohydrates, (e.g., glucose, sucrose, lactose,fructose, maltose, molasses, starch and cellulose), oils and fats,(e.g., soya oil, sunflower oil, groundnut oil and coconut fat), fattyacids (e.g., palmitic acid, stearic acid and linoleic acid), alcohols(e.g., glycerol and ethanol), and organic acids (e.g., acetic acid) maybe used as the carbon source. These substances may be used individuallyor as a mixture.

[0112] Organic nitrogen-containing compounds (e.g., peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea) or inorganic compounds (e.g., ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate)may be used as the nitrogen source. These substances may be usedindividually or as a mixture.

[0113] The phosphorus source may be phosphoric acid, potassiumdihydrogen phosphate or dipotassium hydrogen phosphate (or thecorresponding sodium-containing salts). Furthermore, the culture mediummust comprise salts of metals (e.g., magnesium sulfate or iron sulfate),which are necessary for growth. Finally, essential growth substances,such as amino acids and vitamins, may be used in addition to theabove-mentioned substances. Moreover, suitable precursors may be addedto the culture medium. The starting substances mentioned may be added tothe culture in the form of a single batch, or may be added in a suitablemanner during fermentation.

[0114] Basic compounds (e.g., sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia) or acid compounds (e.g., phosphoric acid orsulfuric acid) may be added in a suitable manner to control the pH ofthe culture. Fatty acid polyglycol esters may be used to control thedevelopment of foam. In order to maintain the stability of plasmids,suitable substances having a selective action, such as antibiotics, maybe added to the medium. In order to maintain aerobic conditions, oxygenor oxygen-containing gas mixtures, such as air, are introduced into theculture. The temperature of the culture is normally from 20° C. to 45°C., and preferably 25° C. to 40° C. Fermentation is continued until themaximum of the desired product has formed. This objective is normallyreached within 10 hours to 160 hours.

[0115] Methods for the determination of L-amino acids are known from theprior art. The analysis can thus be carried out, for example, by anionexchange chromatography with subsequent ninhydrin derivatization asdescribed by Spackman et al. (Analytical Chemistry, 30, 1190 (1958)) orit can be carried out by reversed phase HPLC as described by Lindroth etal. (Analytical Chemistry 51: 1167-1174 (1979)).

[0116] The process according to the invention is used for the productionof amino acids, in particular L-lysin, by fermentation.

[0117] The amino acids are in general isolated by conventional processesor separated off together with constituents of the fermentation brothand optionally the entire biomass or portions thereof.

[0118] The isolation of plasmid DNA from Escherichia coli and alltechniques of restriction, Klenow and alkaline phosphatase treatmentwere performed as described in Sambrook et al. (Molecular Cloning. ALaboratory Manual, 1989, Cold Spring Harbour Laboratory Press, ColdSpring Harbor, N.Y., USA). Methods for transformation of Escherichiacoli and the composition of the usual nutrient media, such as LB or TYmedium, are also described in this handbook.

[0119] The following microorganism was deposited at the DeutscheSammlung für Mikroorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty:

[0120]Escherichia coli Top10/pCR2.1ccpA2int as DSM 14257.

[0121] Having generally described this invention, a furtherunderstanding can be obtained by reference to certain specific exampleswhich are provided herein for purposes of illustration only, and are notintended to be limiting unless otherwise specified.

EXAMPLES Example 1 Preparation of a Genomic Cosmid Gene Library From C.Glutamicum ATCC 13032

[0122] Chromosomal DNA from C. glutamicum ATCC 13032 was isolated asdescribed by Tauch et al. (1995, Plasmid 33:168-179) and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Code no. 27-0913-02). The DNAfragments were dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP, Codeno. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al.(1987), Proceedings of the National Academy of Sciences, USA84:2160-2164), obtained from Stratagene (La Jolla, USA, ProductDescription SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,Product Description XbaI, Code no. 27-0948-02) and likewisedephosphorylated with shrimp alkaline phosphatase.

[0123] The cosmid DNA was then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04). The cosmid DNA so treated was mixed with the treatedATCC13032 DNA and the batch was treated with T4 DNA ligase (AmershamPharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Codeno.27-0870-04). The ligation mixture was then packed in phages with theaid of Gigapack II XL Packing Extract (Stratagene, La Jolla, USA,Product Description Gigapack II XL Packing Extract, Code no. 200217).

[0124] For infection of the E. coli strain NM554 (Raleigh et al. 1988,Nucleic Acid Res. 16:1563-1575) the cells were taken up in 10 mM MgSO₄and mixed with an aliquot of the phage suspension. The infection andtitering of the cosmid library were carried out as described by Sambrooket al. (1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor), the cells being plated out on LB agar (Lennox, 1955, Virology,1:190) containing 100 mg/l ampicillin. After incubation overnight at 37°C., recombinant individual clones were selected.

Example 2 Isolation and Sequencing of the CcpA2 Gene

[0125] The cosmid DNA of an individual colony was isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's instructions and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNAfragments were dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP,Product No. 1758250). After separation by gel electrophoresis, thecosmid fragments ranging from 1500 to 2000 bp were isolated with theQiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

[0126] The DNA of the sequencing vector pZero-1, obtained fromInvitrogen (Groningen, The Netherlands, Product Description ZeroBackground Cloning Kit, Product No. K2500-01) was cleaved with therestriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, ProductDescription BamHI, Product No. 27-0868-04). The ligation of the cosmidfragments in the sequencing vector pZero-1 was carried out as describedby Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor), the DNA mixture being incubated overnight with T4 ligase(Pharmacia Biotech, Freiburg, Germany). This ligation mixture was thenelectroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7)into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of theNational Academy of Sciences, U.S.A., 87:4645-4649) and plated out on LBagar (Lennox, 1955, Virology, 1:190) with 50 mg/l zeocin.

[0127] Plasmid preparation of the recombinant clones was carried outwith the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany).DNA sequencing was administered by the dideoxy chain terminationmethodology of Sanger et al. (1977, Proceedings of the NationalAcademies of Sciences, U.S.A., 74:5463-5467) with modificationsaccording to Zimmermann et al. (1990, Nucleic Acids Research, 18:1067).The “RR dRhodamin Terminator Cycle Sequencing Kit” from PE AppliedBiosystems(Product No. 403044, Weiterstadt, Germany) was used.Separation by gel electrophoresis and analysis of the sequencingreaction were carried out in a “Rotiphoresis NFAcrylamide/Bisacrylamide” Gel (29:1) (Product No. A124.1, Roth,Karlsruhe, Germany) with the “ABI Prism 377” sequencer from PE AppliedBiosystems (Weiterstadt, Germany).

[0128] The raw sequence data obtained were then processed using theStaden program package (1986, Nucleic Acids Research, 14:217-231)version 97-0. The individual sequences of the pZero1 derivatives wereassembled to a continuous contig. The computer-assisted coding regionanalyses were prepared with the XNIP program (Staden, 1986, NucleicAcids Research, 14:217-231). Further analyses were carried out with the“BLAST search program” (Altschul et al., 1997, Nucleic Acids Research,25:3389-3402) against the non-redundant databank of the “National Centerfor Biotechnology Information” (NCBI, Bethesda, Md., USA).

[0129] The resulting nucleotide sequence is shown in SEQ ID No. 1.Analysis of this nucleotide sequence revealed an open reading frame of1041 bp, which was designated the ccpA2 gene. The ccpA2 gene codes for apolypeptide of 346 anino acids (SEQ ID No. 2).

Example 3 Preparation of an Integration Vector for IntegrationMutagenesis of the CcpA2 Gene

[0130] From the C. glutamicum ATCC strain 13032, chromosomal DNA wasisolated by the method of Eikmanns et al. (Microbiology 140: 1817-1828(1994)). On the basis of the sequence of the ccpA2 gene known for C.glutamicum from Example 2, the following oligonucleotides wereobjectively designed for the polymerase chain reaction: ccpA2intA (SEQID NO. 3): 5′AGA GCT GCT TGG TCA GAC TT 3′ ccpA2intB (SEQ ID NO. 4):5′ATC CAG ATT CTT GGC GGT AG 3′

[0131] The primers shown were synthesized by MWG Biotech (Ebersberg,Germany) and the PCR reaction was carried out by the standard PCR methodof Innis et al. (PCR protocols. A guide to methods and applications,1990, Academic Press) with Pwo-Polymerase from Boehringer. With the aidof the polymerase chain reaction, an 322 bp internal fragment of theccpA2 gene (SEQ ID No. 1) was isolated.

[0132] The amplified DNA fragment was ligated into the vectorpCR2.1-TOPO (Mead at al. (1991), Bio/Technology 9:657-663) with the TOPOTA Cloning Kit from Invitrogen Corporation (Carlsbad, Calif., USA;Catalogue Number K4500-01).

[0133] The E. coli strain TOP10F was then transformed with the ligationbatch (Hanahan, Ind.: DNA cloning. A practical approach. Vol. 1,IRL-Press, Oxford, Washington D.C., USA 1985). Selection forplasmid-carrying cells was made by plating out the transformation batchon LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual.2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), which had been supplemented with 25 mg/l kanamycin. PlasmidDNA was isolated from a transformant with the aid of the QIAprep SpinMiniprep Kit from Qiagen and tested by restriction with the restrictionenzyme EcoRI and subsequent agarose gel electrophoresis (0.8%). Theplasmid was designated pCR2.1ccpA2int (FIG. 1). The abbreviations anddesignations used in FIG. 1 have the following meaning.

[0134] KmR: Kanamycin resistance gene

[0135] EcoRI: Cleavage site of the restriction enzyme EcoRI

[0136] HindIII: Cleavage site of the restriction enzyme HindIII

[0137] SacI: Cleavage site of the restriction enzyme SacI

[0138] PstI: Cleavage site of the restriction enzyme PstI

[0139] ccpA2int: Internal fragment of the ccpA2 gene

[0140] ColE1 ori: Replication origin of the plasmid ColE1

Example 4 Integration Mutagenesis of the CcpA2 Gene in the LysineProducer DSM 5715

[0141] The vector pCR2.1ccpA2int mentioned in Example 3 was introducedinto C. glutamicum DSM 5715 (EP 435 132) by the electroporation methodof Tauch et al. (FEMS Microbiological Letters, 123:343-347 (1994)).Strain DSM 5715 is an AEC-resistant lysine producer. The vectorpCR2.1ccpA2int is unable to replicate independently in DSM 5715 and isretained in the cell only if it has been integrated into the chromosomeof DSM 5715. Selection of clones with pCR2.1ccpA2int integrated into thechromosome was performed by plating out the electroporation batch on LBagar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^(nd)Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),which had been supplemented with 15 mg/l kanamycin.

[0142] For detection of the integration, the ccpA2int fragment waslabelled with the Dig hybridization kit from Boehringer by the method of“The DIG System Users Guide for Filter Hybridization” of BoehringerMannheim GmbH (Mannheim, Germany, 1993). Chromosomal DNA of a potentialintegrant was isolated by the method of Eikmanns et al. (Microbiology140: 1817-1828 (1994)) and in each case cleaved with the restrictionenzymes SalI, SacI and HindIII. The fragments formed were separated byagarose gel electrophoresis and hybridized at 68° C. with the Dighybridization kit from Boehringer. The plasmid pCR2.1ccpA2int mentionedin Example 3 had been inserted into the chromosome of DSM 5715 withinthe chromosomal ccpA2 gene. The strain was called DSM5715::pCR2.1ccpA2int.

Example 5 Preparation of L-Lysine

[0143] The C. glutamicum strain DSM 5715::pCR2.1ccpA2int obtained inExample 4 was cultured in a nutrient medium suitable for the productionof L-lysine by fermentation, and the L-lysine content in the culturesupernatant was determined.

[0144] To that end, the strain was first incubated on an agar plate withthe corresponding antibiotic (brain-heart agar with 25 mg/l kanamycin)for 24 hours at 33° C. A pre-culture was inoculated (10 ml medium in a100 ml conical flask). The complete CgIII medium was used as the mediumfor the pre-culture starting from this agar plate culture. Cg III MediumNaCl 2.5 g/l Bacto-Peptone  10 g/l Bacto-Yeast extract  10 g/l Glucose(autoclaved separately) 2% (w/v) The pH was brought to pH 7.4

[0145] Kanamycin (25 mg/l) was added to the pre-culture medium. Thepre-culture was then incubated for 16 hours at 33° C. at 240 rpm on ashaker. A main culture was inoculated from this pre-culture so that theinitial OD (660 nm) of the main culture was 0.1 OD. MM medium was usedfor the main culture. MM Medium CSL (corn steep liquor)   5 g/l MOPS(morpholinopropanesulfonic acid)  20 g/l Glucose (autoclaved separately) 50 g/l Salts: (NH₄)₂SO₄  25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7H₂O 1.0 g/lCaCl₂ * 2H₂O  10 mg/l FeSO₄ * 7H₂O  10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin(sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2 mg/lLeucine (sterile-filtered) 0.1 g/l CaCO₃  25 g/l

[0146] CSL, MOPS and the salt solution are adjusted to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions arethen added, as well as the dry, autoclaved CaCO₃.

[0147] Cell growth was carried out in a 10 ml volume in a 100 ml conicalflask with baffles. Kanamycin (25 mg/l) was added. Cell growth wascarried out at 33° C. and 80% atmospheric humidity.

[0148] After 72 hours, the OD was determined at a measurement wavelengthof 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). Theamount of L-lysine formed was determined with an amino acid analyzerfrom Eppendorf-BioTronik (Hamburg, Germany) by ion-exchangechromatography and post-column derivatization with ninhydrin detection.TABLE 1 OD Lysine HCl Strain (660 nm) g/l DSM 5715 7.9 13.53 DSM5715::pCR2.1ccpA2int 8.1 14.94

[0149] Obviously, numerous modifications and variations on the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1 4 1 1500 DNA Corynebacterium glutamicum CDS (241)..(1278) 1 caagggttgtggatgaacct accccgtgag actccataag taccccatac tttgcccgac 60 gtctcatgaagtgcggtttt aaatcattag ccttcttatt cacttcccgg gatcaccacc 120 cccacttcctacccctgttg ccaacatcgc cttgcacgta ataggttaaa acacaagtga 180 atgtaatcgtttgcagcaat cgattacata aaggtagata atgagataaa gcgaggcgct 240 atg gcg acggaa aaa ttc cga ccg act ctt aaa gat gtc gct cgt caa 288 Met Ala Thr GluLys Phe Arg Pro Thr Leu Lys Asp Val Ala Arg Gln 1 5 10 15 gca ggt gtctcc atc gcc aca gca tca cga gca cta gcg gat aat ccg 336 Ala Gly Val SerIle Ala Thr Ala Ser Arg Ala Leu Ala Asp Asn Pro 20 25 30 gcg gtt gct gcatcg act cgt gaa aga atc caa caa tta gcc tct gat 384 Ala Val Ala Ala SerThr Arg Glu Arg Ile Gln Gln Leu Ala Ser Asp 35 40 45 ctg ggt tac cgg gccaat gct caa gct cgt gcg ctt cgc agt tct cgc 432 Leu Gly Tyr Arg Ala AsnAla Gln Ala Arg Ala Leu Arg Ser Ser Arg 50 55 60 agc aac acc att ggt gtgatt gtt ccc agt ttg att aac cat tac ttc 480 Ser Asn Thr Ile Gly Val IleVal Pro Ser Leu Ile Asn His Tyr Phe 65 70 75 80 gcc gca atg gtt act gaaatt caa agc acc gcc agc aaa gct gga ctt 528 Ala Ala Met Val Thr Glu IleGln Ser Thr Ala Ser Lys Ala Gly Leu 85 90 95 gcc acg att atc acc aac agcaat gaa gat gcg acc act atg tct ggg 576 Ala Thr Ile Ile Thr Asn Ser AsnGlu Asp Ala Thr Thr Met Ser Gly 100 105 110 tct ttg gag ttt ctc acc tcgcat ggt gtc gat gga atc atc tgc gta 624 Ser Leu Glu Phe Leu Thr Ser HisGly Val Asp Gly Ile Ile Cys Val 115 120 125 cct aat gag gaa tgc gcg aatcaa cta gag gac ttg cag aag caa gga 672 Pro Asn Glu Glu Cys Ala Asn GlnLeu Glu Asp Leu Gln Lys Gln Gly 130 135 140 atg cca gtg gtg ttg gtt gaccga gag ctt cca gga gac tcc acc atc 720 Met Pro Val Val Leu Val Asp ArgGlu Leu Pro Gly Asp Ser Thr Ile 145 150 155 160 cca acg gcg acc tct aacccc caa cca gga atc gcc gca gca gta gaa 768 Pro Thr Ala Thr Ser Asn ProGln Pro Gly Ile Ala Ala Ala Val Glu 165 170 175 ctc ctg gct cac aac aacgcg ttg ccg att ggt tac ctc tca ggt ccc 816 Leu Leu Ala His Asn Asn AlaLeu Pro Ile Gly Tyr Leu Ser Gly Pro 180 185 190 atg gac acc tca aca ggtaga gag cga tta gag gat ttc aaa gca gcc 864 Met Asp Thr Ser Thr Gly ArgGlu Arg Leu Glu Asp Phe Lys Ala Ala 195 200 205 tgc gcc aac tcc aaa attggc gaa cag ctc gtt ttt ctg ggt ggg tac 912 Cys Ala Asn Ser Lys Ile GlyGlu Gln Leu Val Phe Leu Gly Gly Tyr 210 215 220 gaa caa agc gtt gga tttgaa ggc gct acg aaa ttg ctc gat caa gga 960 Glu Gln Ser Val Gly Phe GluGly Ala Thr Lys Leu Leu Asp Gln Gly 225 230 235 240 gct aaa act ctt tttgcc ggc gat tct atg atg acg atc ggt gtc att 1008 Ala Lys Thr Leu Phe AlaGly Asp Ser Met Met Thr Ile Gly Val Ile 245 250 255 gaa gcc tgc cat aaggct ggt ttg gtt atc ggc aag gat gtc agc gtg 1056 Glu Ala Cys His Lys AlaGly Leu Val Ile Gly Lys Asp Val Ser Val 260 265 270 att ggt ttt gat acacat ccg ctt ttt gcc ctg caa cct cat ccg ttg 1104 Ile Gly Phe Asp Thr HisPro Leu Phe Ala Leu Gln Pro His Pro Leu 275 280 285 aca gtg att gat caaaat gta gaa caa cta gcc caa cga gca gtg tct 1152 Thr Val Ile Asp Gln AsnVal Glu Gln Leu Ala Gln Arg Ala Val Ser 290 295 300 atc ctc acc gaa ttaatt gca ggc acg gta cct agc gtg acg aaa act 1200 Ile Leu Thr Glu Leu IleAla Gly Thr Val Pro Ser Val Thr Lys Thr 305 310 315 320 acg atc ccc actgcc ctt att cat cgt gaa tca atc atc aac tcc act 1248 Thr Ile Pro Thr AlaLeu Ile His Arg Glu Ser Ile Ile Asn Ser Thr 325 330 335 tta agg aag aaggat gga ctc ccc aat gag taactcaacc ggtaccgaca 1298 Leu Arg Lys Lys AspGly Leu Pro Asn Glu 340 345 ttgtcgttgt cggatccatc aatgccgatc tcaccgcaaaagttcaacgc caccctgaac 1358 ctggagaaac cctcctgggt agcggcggca cagtgagtgctggtggcaaa ggcgccaacc 1418 aagctgtggc ggcagcgcaa ttaggtgcca aagtcaccatgatcggtgcg gtcggaaccg 1478 atcaaatggc tggcgaggcg ct 1500 2 346 PRTCorynebacterium glutamicum 2 Met Ala Thr Glu Lys Phe Arg Pro Thr Leu LysAsp Val Ala Arg Gln 1 5 10 15 Ala Gly Val Ser Ile Ala Thr Ala Ser ArgAla Leu Ala Asp Asn Pro 20 25 30 Ala Val Ala Ala Ser Thr Arg Glu Arg IleGln Gln Leu Ala Ser Asp 35 40 45 Leu Gly Tyr Arg Ala Asn Ala Gln Ala ArgAla Leu Arg Ser Ser Arg 50 55 60 Ser Asn Thr Ile Gly Val Ile Val Pro SerLeu Ile Asn His Tyr Phe 65 70 75 80 Ala Ala Met Val Thr Glu Ile Gln SerThr Ala Ser Lys Ala Gly Leu 85 90 95 Ala Thr Ile Ile Thr Asn Ser Asn GluAsp Ala Thr Thr Met Ser Gly 100 105 110 Ser Leu Glu Phe Leu Thr Ser HisGly Val Asp Gly Ile Ile Cys Val 115 120 125 Pro Asn Glu Glu Cys Ala AsnGln Leu Glu Asp Leu Gln Lys Gln Gly 130 135 140 Met Pro Val Val Leu ValAsp Arg Glu Leu Pro Gly Asp Ser Thr Ile 145 150 155 160 Pro Thr Ala ThrSer Asn Pro Gln Pro Gly Ile Ala Ala Ala Val Glu 165 170 175 Leu Leu AlaHis Asn Asn Ala Leu Pro Ile Gly Tyr Leu Ser Gly Pro 180 185 190 Met AspThr Ser Thr Gly Arg Glu Arg Leu Glu Asp Phe Lys Ala Ala 195 200 205 CysAla Asn Ser Lys Ile Gly Glu Gln Leu Val Phe Leu Gly Gly Tyr 210 215 220Glu Gln Ser Val Gly Phe Glu Gly Ala Thr Lys Leu Leu Asp Gln Gly 225 230235 240 Ala Lys Thr Leu Phe Ala Gly Asp Ser Met Met Thr Ile Gly Val Ile245 250 255 Glu Ala Cys His Lys Ala Gly Leu Val Ile Gly Lys Asp Val SerVal 260 265 270 Ile Gly Phe Asp Thr His Pro Leu Phe Ala Leu Gln Pro HisPro Leu 275 280 285 Thr Val Ile Asp Gln Asn Val Glu Gln Leu Ala Gln ArgAla Val Ser 290 295 300 Ile Leu Thr Glu Leu Ile Ala Gly Thr Val Pro SerVal Thr Lys Thr 305 310 315 320 Thr Ile Pro Thr Ala Leu Ile His Arg GluSer Ile Ile Asn Ser Thr 325 330 335 Leu Arg Lys Lys Asp Gly Leu Pro AsnGlu 340 345 3 20 DNA Artificial Sequence Synthetic DNA 3 agagctgcttggtcagactt 20 4 20 DNA Artificial Sequence Synthetic DNA 4 atccagattcttggcggtag 20

1-29. (Canceled)
 30. A process for producing L-amino acids comprising culturing a bacterial cell in a medium suitable for producing L-amino acids, wherein said bacterial cell comprises an attenuated ccpA2 gene.
 31. The process of claim 30, wherein said bacterial cell is a Coryneform bacterium or Brevibacterim.
 32. The process of claim 31, wherein said bacterial cell is selected from the group consisting of Coryneform glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Brevibacterium favum, Brevibacterium lactofermentum, and Brevibacterium divaricatum.
 33. The process of claim 30, wherein said ccpA2 gene comprises the polynucleotide sequence of SEQ ID NO:
 1. 34. The process of claim 30, wherein said L-amino acid is L-lysine. 35-37. (Canceled) 